This invention relates to the separation of different types of particulate matter in a liquid medium using an ultrasonic standing wave propagated through the medium. It relates particularly, although not exclusively, to a method and a means for chromatography.
In one of its main aspects, the invention is concerned with the separation of biological particles, which term is used here to include a range of particulate matter from macromolecules --e.g. globular proteins --through viruses, bacteria and yeasts, to tissue cells --e.g. plant cells, animal cells and aggregates --but it can also be employed on many finely divided inorganic and organic materials, including siliceous minerals such as clays.
In chemical chromatography, the isolation of chemical components from a mixture for their identification is achieved relying on very small quantities of any one component. That even very complex mixtures can be represented safely by a small sample, so that the column is not overloaded, is due to the uniformity of any given molecular species, individual molecules differing only in features such as isomeric form and isotopic composition to which the separation process is quite insensitive.
It has been proposed in U.S. Pat. No. 4 280 823 to provide a chromatographic column to analyse a sample of red blood cells which is entrained in a gas flow through the column while an ultrasonic transducer at one end of the column directs its output onto a reflector at the opposite end, its frequency and its distance from the reflector being so matched that a standing wave is produced by the interaction of the emitted and reflected waves. It is described in that disclosure how the nodes of the standing wave can function in the same way as a series of filter plates of a chemical chromatograph to promote separation of the constituents of the sample as it moves along the column.
However, biological particles such as cells are much less uniform, individual members of a group differing in size, age, metabolic state and so forth. Moreover, many of these variations within a group are those to which an acoustic separation is acutely sensitive. There are difficulties therefore in applying chromatographic methods using ultrasonic energy to the analysis of large populations of particles and to the detection of fine distinctions of various groups by having each represented by adequate cell populations.
The method disclosed in U.S. Pat. No. 4 280 823 would have at best a limited utility, because to obtain substantial and sufficiently complete separation of any mixed group of biological particles a very large column length is dictated. But apart from the bulk and cost resulting from any substantial increase in size, there is a limit to the maximum column length that can be employed, owing to the attenuation of an ultrasonic wave that occurs with distance and that restricts the length over which the incident and reflected wave energies are sufficiently well matched to form a predominating standing wave. It may be mentioned here that, apart from this major problem, the method disclosed in U.S. Pat. No. 4 280 823 has further disadvantages because of the difficulty of handling biological particles in a gaseous environment, in particular as regards difficulty of control and prevention of damage to or transformation of the particles.
The problem of separating large populations of particles, particularly biological particles may be even more severe if acoustic energy methods are to be employed for a bulk separation process rather than simply the analysis of a very small sample.
It may be expected, for example, that problems would be encountered if an apparatus such as is described in GB No. 2 089 498A were to be used for the separation of large quantities of particles in a mixed population. In that apparatus a flow of liquid in a conduit passes through a zone in which ultrasonic transducers at opposite sides of the conduit are driven with a controlled phase angle between their driving signals so as to establish a standing wave pattern that moves across the conduit, along the common axis of propagation of the two transducers. Particles carried along by the flow through the conduit enter the standing wave transverse to its axis and the acoustic energy is effective only over a very short distance along the length of the conduit. The extent to which particles can be differently displaced along the standing wave is correspondingly severely limited. This limitation, coupled with the difficulties of achieving the separation of groups of non-uniform particles discussed above, means that the apparatus described in GB No. 2 089 498A would have no application to the separation of biological particles.
It is an object of the present invention to provide a method in which the separation of particle types having different acoustic properties can be more effectively performed.